This invention relates generally to a mammalian expression system, and more particularly, relates to a mammalian expression system capable of generating recombinant proteins not heretofore generated at such high levels due to the non-secretor nature of the gene. The recombinant proteins are expressed in culture medium as well as in mammalian cells.
The introduction of the first-generation hepatitis C virus (HCV) enzyme immunoassays (EIAs) (HCV 1.0 EIAs) as screening assays in 1989 and second-generation HCV EIAs in 1992 (HCV 2.0 EIAs) has dramatically reduced the incidence of post-transfusion HCV (PT-HCV) infection in those countries where routine screening of donated blood products is performed. Antibodies to HCV are detected using recombinant proteins derived from the core, NS3 (viral protease) and NS4 (function unknown) genes of the virus. HCV third-generation EIAs (HCV 3.0 EIAs) which include an additional antigen from the NS5 region (containing the viral polymerase and a second unknown function) now are available and in use in several countries. HCV envelope antigens have not been used in these assays.
Difficulties in the expression and purification of the putative HCV viral envelope proteins (E1, E2) have prevented detailed research and possible incorporation of these proteins as targets in blood screening assays. There may be several reasons for the difficulties encountered in getting a cell to synthesize a heterologous protein and subsequently, to detect and recover the protein. For example, the heterologous gene may not be efficiently transcribed into messenger RNA (mRNA). Also, the mRNA may be unstable and degrade prior to translation into the protein. In addition, the ribosome binding site (RBS) present on the mRNA may only poorly initiate translation. The heterologous protein produced may be unstable in the cell or it may be toxic to the cell. If no antibodies to the protein are available or if there is no other way to assay for the protein, it may be difficult to detect the synthesized protein. Lastly, even if the protein is produced, it may be difficult to purify.
Fusion systems provide a means of solving many of the aforementioned problems. The "carrier" portion of the hybrid gene, typically found on the 5' end of the gene, provides the regulatory regions for transcription and translation as well as providing the genetic code for a peptide which facilitates detection (Shuman et al., J. Biol. Chem. 255:168 [1980]) and/or purification (Moks et al., Bio/Technology 5:379 [1987]). Frequently, potential proteolytic cleavage sites are engineered into the fusion protein to allow for the removal of the homologous peptide portion (de Geus et al., Nucleic Acids Res. 15:3743 [1987]; Nambiar et al., Eur. J. Biochem. 163:67 [1987]; Imai et al., J. Biochem. 100:425 [1986]).
When selecting a carrier gene for a fusion system, in addition to detectability and ease of purification, it would be extremely advantageous to start with a highly expressed gene. Expression is the result of not only efficient transcription and translation but also protein stability and benignity (the protein must not harm or inhibit the cell host). Such expression is advantageous because it can enable the production of such fusion proteins for use in assays. In genes where such expression is not possible, it would be advantageous to provide a system whereby a non-secretor gene can secrete, or express, protein in sufficient amounts to be useful in commercial assays or for other purposes such as for vaccine production.